Treatment of hepatic cirrhosis

ABSTRACT

A composition for treating hepatic fibrosis and a method of using and manufacturing the composition are provided. The composition includes a quinazolinone derivative, preferably Halofuginone.

[0001] This is a Continuation Application of U.S. patent applicationSer. No. 08/862,382, filed on May 23, 1997, now abandoned, and aContinuation-in-Part Application of U.S. patent application Ser. No.09/229,894, filed on Jan. 14, 1999, currently pending.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to the treatment of hepaticcirrhosis and, in particular, to the treatment of hepatic cirrhosis withquinazolinone derivatives such as Halofuginone.

[0003] Hepatic cirrhosis has a number of causes, including hepaticfibrosis caused by chronic alcoholism, malnutrition, hemochromatosis,passive congestion, hypercholesterolemia, exposure to hepatotoxicchemical substances, exposure to drugs, immune reactions, geneticallydetermined sensitivities to certain substances as seen with copper inWilson's disease and infections such as viral hepatitis, syphilis andvarious parasitic infections including, but not limited to,Schistosomiasis mansoni and S. japonica. For reasons given in greaterdetail below, the disease is currently incurable and frequently fatal.

[0004] The pathogenesis of hepatic cirrhosis progresses in a number ofstages. First, an enlarged liver is seen with various fatty changes.Next, overt fibrosis is evident with a concomitant decrease in liverfunction. Finally, atrophy of the liver begins, with a correspondingreduction in the size and functionality of the liver. Necrosis of theliver can be seen at any stage, but is particularly pronounced by latestage cirrhosis. Microscopically, a complete disruption of the normalarchitecture of the liver is evident.

[0005] Outside of the liver, other pathological changes become evidentas cirrhosis progresses. Portal circulation is reduced as fibrotictissue is formed in the liver, further reducing liver functionality.This reduced circulation causes an increase in collateral venouscirculation, particularly in the esophagus. These esophageal bloodvessels can rupture, causing fatal hemorrhage. Thus, cirrhosis is anentire pathological process with effects that are not limited to theliver, although the root causes can be found in specific pathologicalchanges to the liver itself.

[0006] One necessary step in the pathogenesis of hepatic cirrhosis isthe formation of fibrotic tissue in the liver. Hepatic fibrosis is afeature of most chronic liver diseases, not just cirrhosis [S. L.Friedman, New Eng. J. Med., 328:1828-35, 1993]. In hepatic fibrosis,connective tissue accumulates in the liver, replacing normal hepaticparenchymal tissue, and reducing liver functionality. The fibrotictissue replaces more complex normal liver tissue in a pathologicalprocess which reduces the amount of liver tissue available for normalfunctions, such as the removal of toxic substances from the blood, andwhich progressively disrupts intrahepatic blood flow. The formation offibrotic tissue in the liver is characterized by the deposition ofabnormally large amounts of extracellular matrix components, includingat least five types of collagen, in particular collagen types I, III,and IV, as well as other matrix proteins [L. Ala-Kokko, Biochem. J.,244:75-9, 1987].

[0007] The synthesis of collagen is also involved in a number of otherpathological conditions. For example, clinical conditions and disordersassociated with primary or secondary fibrosis, such as systemicsclerosis, graft-versus-host disease (GVHD), lung fibrosis and a largevariety of autoimmune disorders, are distinguished by excessiveproduction of connective tissue, which results in the destruction ofnormal tissue architecture and function. These diseases can best beinterpreted in terms of perturbations in cellular functions, a majormanifestation of which is excessive collagen synthesis and deposition.The crucial role of collagen in fibrosis has prompted attempts todevelop drugs that inhibit its accumulation [K. I. Kivirikko, Annals ofMedicine, Vol. 25, pp. 113-126 (1993)].

[0008] Such drugs can act by modulating the synthesis of the procollagenpolypeptide chains, or by inhibiting specific post-translational events,which will lead either to reduced formation of extra-cellular collagenfibers or to an accumulation of fibers with altered properties.Unfortunately, only a few inhibitors of collagen synthesis areavailable, despite the importance of this protein in sustaining tissueintegrity and its involvement in various disorders.

[0009] For example, cytotoxic drugs have been used in an attempt to slowthe proliferation of collagen-producing fibroblasts [J. A. Casas, etal., Ann. Rhem. Dis., 46: 763, 1987], such as colchicine, which slowscollagen secretion into the extracellular matrix [D. Kershenobich, etal., N. Engl. J. Med., 318:1709, 1988], as well as inhibitors of keycollagen metabolism enzymes [K. Karvonen, et al., J. Biol Chem., 265:8414, 1990; C. J. Cunliffe, et al., J. Med. Chem., 35:2652, 1992].

[0010] Unfortunately, none of these inhibitors are collagen-typespecific. Also, there are serious concerns about the toxic consequencesof interfering with biosynthesis of other vital collagenous molecules,such as Clq in the classical complement pathway, acetylcholine esteraseof the neuro-muscular junction endplate, conglutinin and liversurfactant apoprotein.

[0011] Other drugs which can inhibit collagen synthesis, such asnifedipine and phenytoin, inhibit synthesis of other proteins as well,thereby non-specifically blocking the collagen biosynthetic pathway [T.Salo, et al., J. Oral Pathol. Med., 19: 404, 1990].

[0012] Collagen cross-linking inhibitors, such as β-amino-propionitrile,are also non-specific, although they can serve as useful anti-fibroticagents. Their prolonged use causes lathritic syndrome and interfereswith elastogenesis, since elastin, another fibrous connective tissueprotein, is also cross-linked. In addition, the collagen cross-linkinginhibitory effect is secondary, and collagen overproduction has toprecede its degradation by collagenase. Thus, a type-specific inhibitorof the synthesis of collagen itself is clearly required as ananti-fibrotic agent.

[0013] Such a type-specific collagen synthesis inhibitor is disclosed inU.S. Pat. No. 5,449,678 for the treatment of a fibrotic condition. Thisspecific inhibitor is a composition with a pharmaceutically effectiveamount of a pharmaceutically active compound of a formula:

[0014] wherein:

[0015] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy and lower alkoxy; and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are alsoincluded. Of this group of compounds, Halofuginone has been found to beparticularly effective for such treatment.

[0016] U.S. Pat. No. 5,449,678 discloses that these compounds areeffective in the treatment of fibrotic conditions such as sclerodermaand GVHD. PCT Application No. WO 96/06616 further discloses that thesecompounds are effective in treating restenosis. The two formerconditions are associated with excessive collagen deposition, which canbe inhibited by Halofuginone. Restenosis is characterized by smoothmuscle cell proliferation and extracellular matrix accumulation withinthe lumen of affected blood vessels in response to a vascular injury[Choi et al., Arch. Surg., 130:257-261, 1995]. One hallmark of suchsmooth muscle cell proliferation is a phenotypic alteration, from thenormal contractile phenotype to a synthetic one. Type I collagen hasbeen shown to support such a phenotypic alteration, which can be blockedby Halofuginone [Choi et al., Arch. Surg., 130: 257-261, 1995; U.S. Pat.No. 5,449,678].

[0017] However, the in vitro action of Halofuginone does not alwayspredict its in vivo effects. For example, Halofuginone Inhibits thesynthesis of collagen type I in bone chrondrocytes in vitro, asdemonstrated in U.S. Pat. No. 5,449,678. However, chickens treated withHalofuginone were not reported to have an increased rate of bonebreakage, indicating that the effect is not seen in vivo. Thus, theexact behavior of Halofuginone in vivo cannot always be accuratelypredicted from in vitro studies.

[0018] Furthermore, the ability of Halofuginone or other relatedquinazolinone to block or inhibit pathological processes related tohepatic cirrhosis has not been demonstrated. Other inhibitors ofcollagen synthesis, cross-linking and deposition, such ascorticosteroids, penicillamine, methotrexate and colchicine, have beentested for their therapeutic effect on hepatic fibrosis, but have notproved effective [S. L. Friedman, New Eng. J. Med., 328:1828-35, 1993].Although Halofuginone has been shown to have a specific inhibitoryeffect on the synthesis of type I collagen, such inhibition has not beenotherwise shown to be efficacious in the treatment of hepatic cirrhosis.Indeed, hepatic cirrhosis has a high mortality rate, as currentlyavailable therapeutic options have significant side effects and are notgenerally efficacious in slowing or halting the progression of thefibrosis. Furthermore, many other types of extracellular matrixcomponents are deposited during the pathogenesis of hepatic fibrosis,including at least five types of collagen, in particular collagen typesI, III, and IV, as well as other matrix proteins [L. Ala-Kokko, Biochem.J., 244:75-9, 1987]. Thus, merely inhibiting synthesis of collagen typeI would not necessarily slow or halt the development of hepaticfibrosis.

[0019] Thus, simply administering known in vitro inhibitors of collagensynthesis, deposition and cross-linking in an attempt to treat hepaticcirrhosis is ineffective. Clearly, new treatments for this incurabledisease are required which specifically slow or halt the pathogenesis offibrosis, without non-specific or toxic side effects.

[0020] There is thus a widely recognized need for, and it would behighly advantageous to have, a treatment for liver cirrhosis andfibrosis which inhibits fibrogenesis substantially without undesirablenon-specific or toxic side effects.

SUMMARY OF THE INVENTION

[0021] Unexpectedly, it has been found, as described in the examplesbelow, that Halofuginone can also inhibit the pathophysiological processof hepatic fibrosis in vivo, possibly by inhibiting collagen type Isynthesis, although another mechanism or mechanisms could also beresponsible. While inhibition of collagen type I synthesis is proposedas a plausible mechanism, it is not desired to be limited to a singlemechanism, nor is it necessary since the in vivo data presented belowclearly demonstrate the efficacy of Halofuginone as an inhibitor ofhepatic fibrosis in vivo.

[0022] According to the teachings of the present invention, there isprovided a method for the treatment of hepatic cirrhosis in a subject,comprising the step of administering to the subject a pharmaceuticallyeffective amount of a compound having a formula:

[0023] wherein:

[0024] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy;

[0025] R₂ is a member of the group consisting of hydroxy, acetoxy andlower alkoxy, and

[0026] R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl;

[0027] wherein n is 1 or 2; and pharmaceutically acceptable saltsthereof.

[0028] Preferably, the hepatic cirrhosis is caused by contact with ahepatotoxic chemical substance.

[0029] According to another embodiment of the present invention, thereis provided a method for the treatment of hepatic fibrosis in a subject,the hepatic fibrosis being caused by contact with a hepatotoxic chemicalsubstance, the method comprising the step of administering to thesubject a pharmaceutically effective amount of a compound having aformula:

[0030] wherein:

[0031] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy;

[0032] R₂ is a member of the group consisting of hydroxy, acetoxy andlower alkoxy, and

[0033] R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl;

[0034] wherein n is 1 or 2; and pharmaceutically acceptable saltsthereof.

[0035] According to preferred embodiments of the present invention, thehepatic fibrosis is caused by a factor selected from the groupconsisting of chronic alcoholism, malnutrition, hemochromatosis, passivecongestion, hypercholesterolemia, exposure to poisons or toxins,exposure to drugs, immune reactions, genetically determinedsensitivities to a certain substance and infections.

[0036] More preferably, the hepatic fibrosis is caused by a factorselected from the group consisting of viral hepatitis, syphilis and aparasitic infection.

[0037] Most preferably, said parasitic infection is selected from thegroup consisting of Schistosomiasis mansoni and S. japonica.

[0038] According to yet another embodiment of the present invention,there is provided a method for the treatment of an existing condition ofhepatic cirrhosis in a subject, comprising the step of administering tothe subject a pharmaceutically effective amount of a compound having aformula:

[0039] wherein:

[0040] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy;

[0041] R₂ is a member of the group consisting of hydroxy, acetoxy andlower alkoxy, and

[0042] R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl;

[0043] wherein n is 1 or 2; and pharmaceutically acceptable saltsthereof.

[0044] According to further preferred embodiments of the presentinvention, the compound is preferably Halofuginone. Hereinafter, theterm “Halofuginone” is defined as a compound having a formula:

[0045] and pharmaceutically acceptable salts thereof. The compositionpreferably includes a pharmaceutically acceptable carrier for thecompound.

[0046] According to another embodiment of the present invention, thereis provided a method of manufacturing a medicament for treating hepaticcirrhosis, the hepatic cirrhosis being caused by contact with ahepatotoxic chemical substance, the method including the step of placinga pharmaceutically effective amount of a compound in a pharmaceuticallyacceptable carrier, the compound being a member of a group having aformula:

[0047] wherein:

[0048] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy, and lower alkoxy, and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are alsoincluded.

[0049] According to yet another embodiment of the present invention,there is provided a composition for treating hepatic fibrosis, thehepatic fibrosis being caused by contact with a hepatotoxic chemicalsubstance, the composition including a pharmaceutically effective amountof a compound in combination with a pharmaceutically acceptable carrier,the compound being a member of a group having a formula.

[0050] wherein:

[0051] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy, and lower alkoxy; and R₃ is amember of the group consisting of hydrogen and lower alkenoxy; wherein nis 1 or 2. Pharmaceutically acceptable salts thereof are also included.

[0052] According to the present invention, there is also provided amethod of manufacturing a medicament for treating hepatic fibrosis, thehepatic fibrosis being caused by contact with a hepatotoxic chemicalsubstance, the method including the step of placing a pharmaceuticallyeffective amount of a compound in a pharmaceutically acceptable carrier,the compound being a member of a group having a formula:

[0053] wherein:

[0054] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy, and lower alkoxy; and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are alsoincluded.

[0055] According to still another embodiment of the present invention,there is provided a method for the treatment of hepatic cirrhosis in asubject, with the proviso that the hepatic cirrhosis is not caused bychronic alcoholism, viral hepatitis or an autoimmune condition, themethod comprising the step of administering to the subject apharmaceutically effective amount of a compound having a formula:

[0056] wherein:

[0057] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy and lower alkoxy, and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2; and pharmaceutically acceptable salts thereof.

[0058] According to yet another embodiment of the present invention,there is provided a method for the treatment of hepatic fibrosis in asubject, with the proviso that the hepatic fibrosis is not caused bychronic alcoholism, viral hepatitis or an autoimmune condition, themethod comprising the step of administering to the subject apharmaceutically effective amount of a compound having a formula:

[0059] wherein:

[0060] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy and lower alkoxy, and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2; and pharmaceutically acceptable salts thereof.

[0061] Hereinafter, the term “subject” refers to the human or loweranimal to whom Halofuginone was administered. The term “patient” refersto human subjects. The term “treatment” includes slowing or halting theprogression of hepatic cirrhosis or fibrosis once it has arisen. Thephrase “substantially preventing the genesis” of hepatic cirrhosis orfibrosis is understood to refer to the prevention of the appearance ofclinical or preclinical symptoms of these conditions, including theprevention of those symptoms which are indirectly related to thefibrotic and cirrhotic processes themselves, such as hemorrhage fromesophageal blood vessels.

[0062] Hereinafter, the term “hepatotoxic chemical substance” refers tochemicals which are not normally consumed or introduced into thecirculation, and which have a harmful effect on the subject upon contactwith the subject through introduction into the circulation of thesubject, consumption by the subject or introduction to the subjectthrough another route of administration.

[0063] Although the specific quinazolinone derivative “Halofuginone” isreferred to throughout the specification, it is understood that otherquinazolinone derivatives may be used in its place, these derivativeshaving the formula:

[0064] wherein:

[0065] R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy and lower alkoxy, and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are alsoincluded.

[0066] While the invention will now be described in connection withcertain preferred embodiments in the following figures and examples sothat aspects thereof may be more fully understood and appreciated, it isnot intended to limit the invention to these particular embodiments. Onthe contrary, it is intended to cover all alternatives, modificationsand equivalents as may be included within the scope of the invention asdefined by the appended claims. Thus, the following figures and exampleswhich include preferred embodiments will serve to illustrate thepractice of this invention, it being understood that the particularsshown are by way of example and for purposes of illustrative discussionof preferred embodiments of the present invention only, and arepresented in the cause of providing what is believed to be the mostuseful and readily understood description of formulation procedures aswell as of the principles and conceptual aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

[0068] FIGS. 1A-1D illustrate the effect of Halofuginone on collagenα1(I) gene expression in rat liver;

[0069]FIG. 2 illustrates the effect of Halofuginone on hydroxyprolineconcentration in rat liver;

[0070] FIGS. 3A-3D illustrate the effect of Halofuginone on moderatefibrosis in rat liver;

[0071] FIGS. 4A-4F illustrate the effect of Halofuginone for theprevention of TAA-induced liver cirrhosis;

[0072] FIGS. 5A-5F illustrate the effect of Halofuginone for thetreatment of TAA-induced liver cirrhosis;

[0073] FIGS. 6A-6C illustrate the effect of Halofuginone on stellatecells when administered prophylactically for the prevention ofTAA-induced liver cirrhosis; and

[0074] FIGS. 7A-7C illustrate the effect of Halofuginone on stellatecells for the treatment of TAA-induced liver cirrhosis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Unexpectedly, it has been found, as described in the examplesbelow, that Halofuginone can inhibit the pathological process of hepaticcirrhosis in vivo, possibly by inhibiting collagen type I synthesis,although another mechanism or mechanisms could also be responsible.Indeed, irrespective of the specific mechanism, the data presented belowclearly demonstrate the efficacy of Halofuginone in vivo for inhibitionof the pathological progression of hepatic fibrosis.

[0076] Such a finding is unexpected for three reasons. First, thebehavior of Halofuginone in vitro does not exactly correspond to itsbehavior in vivo. This can be demonstrated by the differential effect ofHalofuginone observed with bone chondrocytes in vivo and in vitro.Halofuginone inhibits the synthesis of collagen type I in chrondrocytesin vitro, as demonstrated in U.S. Pat. No. 5,449,678. However, chickenstreated with Halofuginone were not reported to have an increased rate ofbone breakage, indicating that the effect is not seen in vivo. Thus, theexact behavior of Halofuginone in vivo cannot always be accuratelypredicted from in vitro studies.

[0077] Second, other inhibitors of collagen synthesis, deposition andcross-linking have not proved effective for the treatment of hepaticcirrhosis, demonstrating that inhibition of collagen production alone isnot sufficient for determining the success or failure of a treatment forhepatic fibrosis. Thus, the finding that Halofuginone can successfullyinhibit hepatic fibrosis in vivo is both novel and non-obvious.

[0078] Third, Halofuginone has only been shown to be a collagen type Iinhibitor. However, the formation of fibrotic tissue in the liver ischaracterized by the deposition of abnormally large amounts ofextracellular matrix components, including at least five types ofcollagen, in particular collagen types I, III, and IV, as well as othermatrix proteins [L. Ala-Kokko, Biochem. J., 244:75-9, 1987]. Thus, theability of Halofuginone to inhibit collagen type I synthesis anddeposition cannot predict the ability of Halofuginone to slow, reduce orother ameliorate the pathogenesis of hepatic fibrosis.

[0079] Finally, all other prior art references have only taught theefficacy of Halofuginone on cells such as fibroblasts. In the liver, Itocells have been shown to be the source of the extracellular matrixcomponents which are produced during liver fibrosis, so this cell typeis crucial to the pathogenesis of liver fibrosis [S. L. Friedman, NewEng. J. Med., 328:1828-35, 1993]. However, Ito cells are a completelydifferent cell type than fibroblasts. In addition, Halofuginone has beenshown to be surprisingly effective for the prevention of the abnormalexpression of smooth muscle cell characteristics of stellate cells,which are certainly different from those cells previously shown to beinvolved in fibrotic processes. Even if the behavior of Halofuginone oncells of a certain type could be predicted, such a prediction wouldcertainly not be reliable for cells outside of that type. Thus, theeffect of Halofuginone on Ito cells is not predictable from its effecton fibroblasts.

[0080] Thus, nothing in the prior art taught that Halofuginone would beuseful in the treatment of hepatic fibrosis in vivo. Furthermore, theability of Halofuginone, and related compounds, to slow or haltprogression of fibrosis in the liver is both novel and non-obvious. Thedemonstration of such an ability in vivo is particularly unexpected,given the differential responses seen in vitro and in vivo toHalofuginone. Furthermore, the ability of Halofuginone to treat andameliorate an existing liver cirrhotic condition, as demonstrated below,is particularly unexpected, since previously Halofuginone was shown tohave greater efficacy for the prevention of liver cirrhosis throughprophylactic administration.

[0081] The present invention may be more readily understood withreference to the following illustrative examples and figures. It shouldbe noted that although reference is made exclusively to Halofuginone, itis believed that the other quinazolinone derivatives described andclaimed in U.S. Pat. No. 3,320,124, the teachings of which areincorporated herein by reference, have similar properties.

[0082] The present invention is of a treatment for hepatic cirrhosis andfibrosis with quinazolinone-containing compounds such as Halofuginone.Both compositions with specific pharmaceutical formulations and methodsof using these compounds are described below.

[0083] Although the pathogenesis of hepatic cirrhosis is not fullyunderstood, animal models for the disease have been successfullydeveloped. Hepatic fibrosis has been induced in rats by theintraperitoneal injection of dimethylnitrosamine, with a relativelyshort onset of action: within three weeks of administration ofdimethylnitrosamine to rats, hepatic fibrosis was already evident [A. M.Jezequel et al., J. Hepatol., 5:174-81, 1987].

[0084] Dimethylnitrosamine-induced hepatic fibrosis is characterized byincreased deposition of extracellular matrix components, includingvarious types of collagen such as collagen type I. Thus, inhibition offibrosis, as in both dimethylnitrosamine-induced and other types ofhepatic fibrosis, depends upon the slowing or halting of thepathological process leading to the production of fibrotic tissue.

[0085] Therefore, compounds which are intended for the inhibition ofhepatic cirrhosis must be tested in an in vivo model, such as thedimethylnitrosamine model described above, for their ability to slow orhalt the pathological process leading to deposition of fibrotic tissueSuch experiments were conducted for the collagen type I synthesisinhibitor Halofuginone, as described in greater detail in Examples 1 and2 below.

[0086] Furthermore, once demonstrably effective compounds have beendiscovered, specific formulations and routes of administration must beelucidated for maximum efficacy of the treatment. Such formulations androutes of administration must enable the compound to be effectivelyabsorbed and delivered to the desired site of treatment, whileminimizing non-specific side effects caused by systemic distribution ofthe compound. Illustrative examples of these formulations and routes ofadministration for quinazolinone-containing compounds such asHalofuginone are given in Examples 3-5 below.

EXAMPLE 1

[0087] Effect of Halofuginone on Histology and Morphology of Rat Liver

[0088] Histological examination of liver samples from control anddimethylnitrosamine-treated rats revealed that dimethylnitrosamineinduced specific morphological changes in rat liver, including increasedcollagen fiber content. Dimethylnitrosamine is an example of ahepatotoxic chemical substance as previously defined. Halofuginonesubstantially inhibited the occurrence of these morphological changes,resulting in rat liver of more normal appearance.

[0089] The experimental method was as follows. Male Sprague-Dawley ratswere divided into four groups. Two groups were injectedintraperitoneally with 1% dimethylnitrosamine in saline for threeconsecutive days per week for 3 weeks, at a dose of 1 ml/kg body weight.This dosage regimen will induce severe liver fibrosis. The other twogroups of rats, control rats, were injected with saline. One group ofdimethylnitrosamine-treated rats and one control group were fedHalofuginone in the diet at a dose of 5 mg/kg weight of diet, startingthree days before the dimethylnitrosamine injections were administered.At the end of the experimental period, the rats were sacrificed and theliver was removed and weighed.

[0090] Liver samples were taken for histological examination. Briefly,the tissue samples were collected into phosphate-buffered saline (PBS)and fixed overnight in 4% paraformaldehyde in PBS at 4° C. Serial 5 μmsections were prepared after the samples had been dehydrated in gradedethanol solutions, cleared in chloroform and embedded in Paraplast.Differential staining of collagenous and non-collagenous proteins wasperformed with 0.1% Sirius red and 0.1% fast green as a counter-stain inpicric acid. This procedure stains collagen red [Gascon-Barre, M., etal., J. Histochem. Cytochem., 37:377-381, 1989].

[0091] Liver samples were then hybridized with a probe for rat collagenα1(I) expression. For hybridization with the genetic probe, the sectionswere deparafinized in xylene, rehydrated through a graded series ofethanol solutions, rinsed in distilled water for 5 minutes and thenincubated in 2×SSC at 70° C. for 30 minutes. The sections were thenrinsed in distilled water and treated with pronase, 0.125 mg/ml in 50 mMTris-HCl, 5 mM EDTA, pH 7.5, for 10 minutes. After digestion, the slideswere rinsed with distilled water, post-fixed in 10% formalin in PBS andblocked in 0.2% glycine. After blocking, the slides were rinsed indistilled water, rapidly dehydrated through graded ethanol solutions andair-dried for several hours. Before hybridization, the 1600 bp ratcollagen α1(I) insert was cut out from the original plasmid, pUC18, andinserted into the pSafyre plasmid. The sections were then hybridizedwith this probe after digoxigenin-labeling [M. Pines et al., MatrixBiology, 14:765-71, 1996].

[0092]FIG. 1 shows in situ hybridization of a section of rat livertissue with rat collagen α1(I) probe. A low expression of collagen α1(I)gene is seen in liver of control rats (FIG. 1A) or rats givenHalofuginone alone (FIG. 1B). A marked increase in the expression ofcollagen α1(I) gene was seen in the liver of rats givendimethylnitrosamine alone (FIG. 1C). The gene expression was mainly inthe septa surrounding the lobules at the site of sparse collagenoustissue. Rats given both Halofuginone and dimethylnitrosamine show amarked reduction in the expression of collagen α1(I) gene (FIG. 1D), ascompared to rats given dimethylnitrosamine alone. Although this dose ofHalofuginone substantially reduced the increase in rat collagen α1(I)gene expression caused by dimethylnitrosamine, it did not completelyinhibit such expression as traces can be observed (see arrows). However,the substantially reduced rat collagen α1(I) gene expression indicatesthat Halofuginone is effective against the pathological induction ofexpression by dimethylnitrosamine.

[0093] Sections of rat liver tissue were stained with Sirius red todemonstrate collagen content of the tissue, although results are notshown pictorially since the histological samples must be viewed in colorin order to see the effects. Almost no collagen fibers were observed inliver tissue taken from control rats or rats given Halofuginone alone.The livers of the dimethylnitrosamine-treated rats exhibited an increasein collagen content, displaying bundles of collagen surrounding thelobules, resulting in large fibrous septa. The thickening of thesecollagen bundles was markedly reduced in rats given bothdimethylnitrosamine and Halofuginone, again indicating the ability ofHalofuginone to substantially inhibit the pathophysiological process offibrosis induced by dimethylnitrosamine.

[0094] Interestingly, the relatively high dose of dimethylnitrosaminecaused such severe hepatic fibrosis that four out of the sixdimethylnitrosamine-treated rats which were not given Halofuginone haddied by the end of three weeks. By contrast, only one of the six ratsgiven both dimethylnitrosamine and Halofuginone died. Each of the sixrats in the two groups which were not given dimethylnitrosaminesurvived. Thus, Halofuginone alone had no toxicity, yet was able toalmost completely prevent dimethylnitrosamine-induced death.

[0095] Dimethylnitrosamine-induced changes on the gross morphologicallevel were also inhibited by Halofuginone. Rats treated withdimethylnitrosamine alone had significantly lower liver weights (4.5 gand 5.0 g), particularly when compared to control rats and rats givenHalofuginone alone (12±1 g and 11±1.5 g, respectively). Rats given bothHalofuginone and dimethylnitrosamine had liver weights (8.5±1.7 g) thatwere almost twice that of rats given dimethylnitrosamine alone, althoughsomewhat reduced as compared to control rats.

[0096] Thus, Halofuginone was able to prevent the appearance of theeffects of dimethylnitrosamine-induced fibrosis on all levelsnear-elimination of dimethylnitrosamine-induced fatalities, and markedreduction of gross and fine morphological changes caused bydimethylnitrosamine-induced fibrosis. Clearly, the effects ofHalofuginone are both potent and specific for the prevention of themorphological changes produced during the pathological process ofhepatic fibrosis.

EXAMPLE 2

[0097] Effect of Halofuginone on Mild Fibrosis in Rat Liver

[0098] Halofuginone substantially completely prevented milddimethylnitrosamine-induced fibrosis, as demonstrated by the measurementof collagen α1(I) gene expression and hydroxyproline content. Thespecific experimental method used was similar to that of Example 1,except that the dimethylnitrosamine-treated rats were only given 0.25%dimethylnitrosamine in saline, a much lower dose than that given inExample 1 above. Also, the duration of treatment was longer before therats were sacrificed: 4 weeks as opposed to 3 weeks in Example 1.

[0099] The expression of the collagen α1(I) gene was measured asdescribed in Example 1 above. For hydroxyproline analysis, liver sampleswere hydrolyzed for 22 hours at 110° C. with 6 N HCl. Nitrogen wasdetermined after Kjeldahl digestion by the spectrophotometric procedureusing an autoanalyzer as described by Krom [M. D. Krom, Analyst,105:305-16, 1980]. The collagen-unique amino acid hydroxyproline fromthe same hydrolysate was determined by amino acid analysis (Biotronik LC5000, Germany) after post-column derivatization on a cation exchangecolumn (BTC 2710, Biotronik). The results are expressed as thepercentage of collagen in total liver proteins.

[0100] Hydroxyproline is an amino acid which is present in relativelylarge amounts in collagen, and therefore serves as an indicator for theoverall level of collagen in a particular tissue. Thus, as shown in FIG.2, dimethylnitrosamine clearly caused a significant increase inhydroxyproline concentration, and therefore of collagen levels, in thelivers of rats. This increase was completely inhibited by treatment withHalofuginone. However, administration of Halofuginone to rats which werenot given dimethylnitrosamine did not cause any change in hydroxyprolineconcentration. Therefore, the effect of Halofuginone was simply toinhibit the dimethylnitrosamine-induced increase in hydroxyprolineconcentration.

[0101]FIG. 3C demonstrates that such a low dose of dimethylnitrosaminestill caused an increase in collagen α1(I) gene expression, especiallyby cells surrounding the blood vessels. FIG. 3D shows that thisincreased gene expression was abolished by Halofuginone. Again, as inExample 1 above, Halofuginone alone had no effect on collagen α1(I) geneexpression (FIG. 3B), while control rats also had no collagen α1(I) geneexpression (FIG. 3A).

[0102] Thus, clearly Halofuginone completely inhibited the increasedlevels of collagen synthesis induced by dimethylnitrosamine in thelivers of rats. However, Halofuginone alone did not demonstrate any sucheffect in rats, indicating that the effect of Halofuginone is specificfor inhibition of those pathophysiological processes, such as collagensynthesis, which are caused by dimethylnitrosamine-induced fibrosis.Furthermore, Halofuginone was clearly able to substantially completelyabrogate the biochemical and physiological changes caused bydimethylnitrosamine, as demonstrated by both Examples 1 and 2.

EXAMPLE 3

[0103] Inhibition of Fibrosis Induced by Bile Duct Ligation

[0104] In addition to dimethylnitrosamine-induced liver fibrosis, asecond model of liver fibrosis in rats is available. This model reliesupon surgical ligation of the bile duct to induce liver fibrosis, ratherthan requiring the administration of exogeneous substances or toxicchemicals, and has been shown to be a suitable model for studying humanliver cirrhosis [Kountaras, J. et al., Br. J. Exp. Pathol., 65:305-311,1984; Muriel, P. et al., J. Hepatol., 21:95-102, 1994; Muriel P. et al.,J. Appl. Tox., 15:449-453, 1995]. Thus, the particular advantage of thebile duct ligation model is that any protective treatments must directlyprotect the liver from the pathological changes induced by fibrosis,rather than indirectly altering the effects of the exogeneous substancewhich is used to cause liver fibrosis in the animal model. Theexperimental method was as follows.

[0105] Male Wistar rats, weighing 200-250 g, were divided into fourexperimental groups with 3 rats in each group. The first group did nothave bile duct ligation surgery and was not given Halofuginone. Thesecond group did not have bile duct ligation surgery and was givenHalofuginone. It should be noted that all animals in the first twogroups underwent sham operations which included all steps of the actualsurgical procedure, with the exception of the bile duct ligation itself.The third group had bile duct ligation surgery and was not givenHalofuginone. The fourth group had bile duct ligation surgery and wasgiven Halofuginone. The actual surgical procedure was essentiallysimilar to that reported in the literature [Kountaras, J. et al., Br. J.Exp. Pathol., 65:305-311, 1984].

[0106] All animals were given drinking water ad libitum. Rats which weregiven Halofuginone were fed Halofuginone in the normal rat diet at aconcentration of 5 mg per kg diet weight for one week before surgery andfor the duration of the experimental period, which was either 3 or 7days after the surgical operation. Rats were sacrificed at the end ofthe experimental period. Both collagen content (through Sirius redstaining) and collagen α1(I) gene expression were measured as describedabove in Example 1. In addition, serum alkaline phosphatase, alanineaminotransferase and aspartate aminotransferase levels were measuredcolorimetrically by a Hitachi Auto-analyzer System ofBoerringher-Mannheim. Results are as follows.

[0107] No collagen synthesis was observed in rats which underwent a shamoperation. Furthermore, these rats did not show any increase in bodyweight or liver weight, or any altered liver histology. Finally, theserats did not show any changes in the levels of the enzymes alkalinephosphatase, alanine aminotransferase or aspartate aminotransferaseeither 3 or 7 days after the operation, regardless of whetherHalofuginone was administered.

[0108] By contrast, elevated levels of all three enzymes were observedin rats which underwent bile duct ligation in both theHalofuginone-treated and untreated groups. These elevated levels arecharacteristic markers for the pathological process of liver fibrosisand cirrhosis. However, rats which were fed Halofuginone had lowerlevels of these enzymes than rats which were not. Specifically, ratswhich were not given Halofuginone had 56% higher alanineaminotransferase, 257% alkaline phosphatase and 15% higher aspartateaminotransferase levels than rats which were fed Halofuginone. Thus,clearly Halofuginone reduced the extent of elevated enzyme levels inrats which underwent bile duct ligation.

[0109] Furthermore, Halofuginone significantly reduced the bile ductligation-induced increased in collagen synthesis and collagen α1(I) geneexpression, when rats which underwent bile duct ligation and which werefed Halofuginone were compared to rats which only underwent bile ductligation. Thus, Halofuginone clearly was able to inhibit the process ofliver fibrosis in the model of bile duct ligation-induced fibrosis inrats.

EXAMPLE 4

[0110] Treatment of Liver Cirrhosis by Halofuginone

[0111] Another model of liver cirrhosis is the chemical induction ofsuch cirrhosis with thioacetamide (TAA) in rats. The administration ofthioacetamide by intra-peritoneal (i.p.) injection induces livercirrhosis, including the deposition of fibrotic tissues and the loss ofliver function. Halofuginone was shown to be effective for both thetreatment of liver cirrhosis after the appearance of cirrhotic symptoms,and for the prevention of liver cirrhosis The latter effect isparticularly surprising since previous medicaments had not beenpreviously shown to be able to treat an existing cirrhotic condition,which is important since the administration of such medicaments to humanpatients is typically performed only after liver cirrhosis has arisen inthe patient. The experimental method was as follows.

[0112] Male Sprague-Dawley rats were divided into three groups. Twogroups were injected intraperitoneally with TAA twice weekly for 12weeks, at a dose of 200 mg/kg body (Muller, A. et al., Exp. Pathol.,34:229-236, 1988; Hori, N. et al., Dig. Dis. Sci., 38:2195-2202, 1993).Such long term administration of TAA results in characteristic lesionswhich demonstrate cirrhosis-like patterns of micronodular cirrhosis inthe liver of the rats. The other group of rats, control rats, wasinjected with saline. One group of TAA-treated rats and one controlgroup were fed Halofuginone in the diet at a dose of 5 ppm. Two separateexperiments were performed, one experiment for demonstrating theefficacy of Halofuginone for the treatment of liver cirrhosis, and oneexperiment for demonstrating the prevention of liver cirrhosis. Again,it should be emphasized that previously only the efficacy of preventionof liver cirrhosis with Halofuginone had been demonstrated, but not thetreatment of existing liver cirrhosis.

[0113] Prevention of Liver Cirrhosis

[0114] For this experiment, rats in group 1 received only Halofuginone(n=4); rats in group 2 received TAA alone without Halofuginone (n=6);and rats in group 3 received TAA with Halofuginone (n=6). Groups 1 and 3received Halofuginone starting three days before the TAA injections wereadministered. At the end of the experimental period, the rats weresacrificed and the liver was removed and weighed.

[0115] Liver samples were taken for histological examination. Briefly,the tissue samples were collected into phosphate-buffered saline (PBS)and fixed overnight in 4% paraformaldehyde in PBS at 4° C. Serial 5 μmsections were prepared after the samples had been dehydrated in gradedethanol solutions, cleared in chloroform and embedded in Paraplast.Differential staining of collagenous and non-collagenous proteins wasperformed with 0.1% Sirius red and 0.1% fast green as a counter-stain inpicric acid. This procedure stains collagen red [Gascon-Barre, M., etal., J. Histochem. Cytochem., 37:377-381, 1989].

[0116] In addition, the livers were scored in a double-blind evaluation,on a scale of 0-4, for the appearance of fibrotic tissue. Previously, anexcellent correlation has been demonstrated between such manualdouble-blind scores and the deposition of collagen as evaluated byhistology, for example.

[0117] Sections of rat liver tissue were stained with Sirius red todemonstrate collagen content of the tissue, as shown in FIGS. 4A-4F.Almost no collagen fibers were observed in liver tissue taken from ratsgiven Halofuginone alone (FIGS. 4A and 4B, representing samples from twodifferent rats). The livers of the TAA-treated rats exhibited anincrease in collagen content, displaying bundles of collagen surroundingthe lobules, resulting in large fibrous septa (FIGS. 4C and 4D,representing samples from two different rats). The thickening of thesecollagen bundles was markedly reduced in rats given both TAA andHalofuginone, again indicating the ability of Halofuginone tosubstantially inhibit the pathophysiological process of fibrosis inducedby TAA (FIGS. 4E and 4F, representing samples from two different rats).

[0118] TAA-induced changes on the gross morphological level were alsoinhibited by Halofuginone, as shown by the manual double blind scores.Rats treated with TAA alone had an average score of 2.16±0.7, whilecontrol rats had a score of 0 and rats given both Halofuginone and TAAhad an average score of 1.25±0.8, which is significantly lower than theaverage score of rats given TAA alone. Thus, no effect of Halofuginoneon body weight or spleen weight was demonstrated (data not shown), whilethe fibrotic scores for liver were reduced by 43%.

[0119] Thus, Halofuginone was able to prevent the appearance of theeffects of TAA-induced fibrosis on all levels, including markedreduction of gross and fine morphological changes caused by TAA-inducedfibrosis.

[0120] Treatment of Liver Cirrhosis

[0121] The experimental method was similar for the treatment of existingliver cirrhosis, as previously described, except that Halofuginone wasnot administered until after the induction of liver cirrhosis bytreatment with TAA for twelve weeks. For this experiment, rats in group1 received only TAA (n=6) and were killed after the initial twelve weekperiod. The remaining rats were divided into two groups, and eitherreceived no further treatment (group 2, n=8), or Halofuginone (group 3,n=8). Rats in groups 2 and 3 were killed four weeks later.

[0122] As previously described, sections of rat liver tissue werestained with Sirius red to demonstrate collagen content of the tissue,as shown in FIGS. 5A-5F. Th livers of the TAA-treated rats exhibited anincrease in collagen content, displaying bundles of collagen surroundingthe lobules, resulting in large fibrous septa (FIGS. 4A and 4B, and 4Cand 4D, representing samples from four different rats). The thickeningof these collagen bundles was markedly reduced in rats given both TAAand Halofuginone, again indicating the ability of Halofuginone tosubstantially inhibit the pathophysiological process of fibrosis inducedby TAA (FIGS. 4E and 4F, representing samples from two different rats).

[0123] TAA-induced changes on the gross morphological level were alsoinhibited by Halofuginone, as shown by the manual double blind scores.Rats treated with TAA alone had an average score of 2.16±0.7 or 3.06±1.7for groups 1 and 2 respectively, while rats given both Halofuginone andTAA had an average score of 1.18±1.6 for group 3, which is significantlylower than the average score of rats given TAA alone and is similar tothe effect of Halofuginone as a prophylactic agent. Thus, the fibroticscores for liver were significantly reduced, even to as great an extentas for rats given Halofuginone before TAA was administered, therebydemonstrating the efficacy of Halofuginone for the treatment of anexisting liver cirrhotic and fibrotic condition.

[0124] Clearly, the effects of Halofuginone are both potent and specificfor the prevention of the morphological changes produced during thepathological process of hepatic fibrosis, both as a treatment forexisting fibrotic and cirrhotic conditions, and as a preventative agent.

EXAMPLE 5

[0125] Effect of Halofuginone on Stellate Cells

[0126] Liver cirrhosis was induced with TAA as previously described. Thetypes of cells injured through the cirrhotic process were then examined,and stellate cells of the liver were found to be targets of theTAA-induced liver cirrhosis. Previously, stellate cells had been shownto produce the excess extracellular matrix components associated withliver cirrhosis and fibrosis (S. L. Friedman, New England J. Med.,328:1828-1835, 1993). These cells are located in the subendothelialspace, between the apical membrane of hepatocytes and the ablumenal sideof the sinusoidal endothelial cells. The intimate association betweenthe stellate cells and the neighboring cell types facilitatesintercellular transport and paracrine stimulation by soluble mediators.In injured liver, these cells acquire features of myofibroblasts andexpress smooth muscle cytoskeletal markers such as smooth muscle actin.The level of smooth muscle actin is greatly increased during liverinjury in regions of fibrous septa formation adjacent to hepatocellularinjury, and is localized within the microfilament bundles of cells. Inthe present experiments, Halofuginone was shown to ameliorate the effectof TAA-induced damage to the stellate cells. The experimental method wasas follows.

[0127] Rats were treated with TAA alone or TAA with Halofuginone aspreviously described in Example 4 for the use of Halofuginone as both aprophylactic treatment and a treatment for existing liver cirrhosis.Tissue samples were also prepared as previously described, except thatrather than undergoing staining for collagen, the samples were incubatedwith smooth muscle actin antibodies in a 1:1000 dilution; with detectionperformed by the Histomouse SP kit according to the instructions of themanufacturer (Zymed Laboratories Inc., San Francisco, Calif., USA).

[0128]FIG. 6 shows the detection of smooth muscle actin in liver tissuetaken from rats in the prevention experiment, while FIG. 7 shows suchdetection in liver tissue taken from rats in the treatment experiment.As shown in FIG. 6A, in samples of liver tissue taken from control rats,smooth muscle actin was only found in cells surrounding the bloodvessels, which is the normal pattern of detection. However, samples ofliver taken from rats after the administration of TAA alone show thatTAA-induced liver cirrhosis causes smooth muscle actin to also beexpressed by stellate cells, found in the septa formed by the depositionof additional extracellular matrix components (FIG. 6B). Such abnormal,pathological expression of smooth muscle actin was prevented by theprophylactic administration of Halofuginone (FIG. 6C).

[0129] Similarly, the administration of TAA alone in the secondexperiment, testing Halofuginone as a treatment of existing livercirrhosis, also resulted in a high level of smooth muscle actinexpression by stellate cells (FIG. 7A), which was maintained 8 weekslater (FIG. 7B). However, the administration of Halofuginone, even afterthe induction of liver cirrhosis, significantly reduced the pathologicalexpression of smooth muscle actin (FIG. 7C). Thus, again Halofuginone isshown to be effective as both a prophylactic agent for preventing livercirrhosis, and for the treatment of existing cirrhosis, at leastpartially in the liver by preventing the pathological expression ofsmooth muscle characteristics by stellate cells.

EXAMPLE 6

[0130] Suitable Formulations for Administration of Halofuginone

[0131] Halofuginone can be administered to a subject in a number ofways, which are well known in the art. Hereinafter, the term “subject”refers to the human or lower animal to whom Halofuginone wasadministered. For example, administration may be done orally, orparenterally, for example by intravenous drip or intraperitoneal,subcutaneous, or intramuscular injection.

[0132] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets,capsules or tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable.

[0133] Formulations for parenteral administration may include but arenot limited to sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

[0134] According to a preferred embodiment of the present invention, oneadditional route of administration of Halofuginone is through a medicaldevice.

[0135] Dosing is dependent on the severity of the symptoms and on theresponsiveness of the subject to Halofuginone. Persons of ordinary skillin the art can easily determine optimum dosages, dosing methodologiesand repetition rates.

EXAMPLE 7

[0136] Method of Treatment of Hepatic Fibrosis and Cirrhosis

[0137] As noted above, Halofuginone has been shown to be an effectiveinhibitor of hepatic fibrosis, a precursor of hepatic cirrhosis. Thefollowing example is an illustration only of a method of treatinghepatic fibrosis and cirrhosis with Halofuginone, and is not intended tobe limiting.

[0138] The method includes the step of administering Halofuginone, in apharmaceutically acceptable carrier as described in Example 4 above, toa subject to be treated. Halofuginone is administered according to aneffective dosing methodology, preferably until a predefined endpoint isreached, such as the absence of further progression of hepatic fibrosisor cirrhosis in the subject, the inhibition of hepatic fibrosis orcirrhosis or the prevention of the formation of hepatic fibrosis orcirrhosis.

[0139] Examples of types of hepatic fibrosis for which such a treatmentwould be effective include, but are not limited to, hepatic fibrosiscaused by chronic alcoholism, malnutrition, hemochromatosis, passivecongestion, hypercholesterolemia, exposure to poisons or toxins,exposure to drugs, immune reactions, genetically determinedsensitivities to certain substances as seen with copper in Wilson'sdisease and infections such as viral hepatitis, syphilis and variousparasitic infections including, but not limited to, Schistosomiasismansoni and S. japonica. Other examples of infections which may causesuch a liver fibrotic condition include, but are not limited to, CMV(cytomegalovirus), EBY (Epstein-Barr virus) and various herpes viruses.Hepatitis may also be caused by autoimmune conditions. Other examples ofconditions which-feature liver fibrosis include, but are not limited to,primary biliary cirrhosis, primary sclerosing cholansitis, chronic liverrejection, cryptogenic liver fibrosis and congenital liver fibrosis.

[0140] The terms “poison” and “toxin” hereinafter refer to substanceswhich are hepatotoxic chemical substances, and therefore which are notnormally consumed or introduced into the circulation and which have aharmful effect on the subject upon contact with the subject throughintroduction into the circulation of the subject, consumption by thesubject or introduction to the subject through another route ofadministration. One non-limiting example of such a hepatotoxic chemicalsubstance is lead.

[0141] In addition, such a treatment would also be effective for hepaticfibrotic conditions of unknown or poorly defined etiology. Preferably,the methods of the present invention for the treatment or prevention ofhepatic cirrhosis or fibrosis include contact with a hepatotoxicchemical substance as the cause for the hepatic fibrosis or cirrhosis.

[0142] Since hepatic fibrosis is a necessary precursor for livercirrhosis, all of these methods can also be used to treat or preventliver cirrhosis, in addition to treating or preventing those conditionscharacterized by liver fibrosis alone.

[0143] Examples of conditions which are characterized by liver fibrosisalone include, but are not limited to, periportal fibrosis,presinusiodal fibrosis and postsinusiodal fibrosis.

EXAMPLE 8

[0144] Method of Manufacture of a Medicament Containing Halofuginone

[0145] The following is an example of a method of manufacturingHalofuginone. First, Halofuginone is synthesized in accordance with goodpharmaceutical manufacturing practice. Examples of methods ofsynthesizing Halofuginone, and related quinazolinone derivatives, aregiven in U.S. Pat. No. 3,338,909. Next, Halofuginone is placed in asuitable pharmaceutical carrier, as described in Example 6 above, againin accordance with good pharmaceutical manufacturing practice.

[0146] It will be appreciated that the above descriptions are intendedonly to serve as examples, and that many other embodiments are possiblewithin the spirit and the scope of the present invention.

What is claimed is:
 1. A method for the treatment of hepatic cirrhosisin a subject, comprising the step of administering to the subject apharmaceutically effective amount of a compound having a formula:

wherein: R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy and lower alkoxy, and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2; and pharmaceutically acceptable salts thereof. 2.The method of claim 1, wherein said compound is Halofuginone.
 3. Themethod of claim 1, wherein said compound further includes apharmaceutically acceptable carrier.
 4. The method of claim 1, whereinthe hepatic cirrhosis is caused by contact with a hepatotoxic chemicalsubstance.
 5. The method of claim 1, wherein the hepatic cirrhosiscaused by a factor selected from the group consisting of chronicalcoholism, malnutrition, hemochromatosis, passive congestion,hypercholesterolemia, exposure to poisons or toxins, exposure to drugs,immune reactions, genetically determined sensitivities to a certainsubstance and infections.
 6. The method of claim 5, wherein the hepaticcirrhosis is caused by a factor selected from the group consisting ofviral hepatitis, syphilis and a parasitic infection.
 7. The method ofclaim 6, wherein said parasitic infection is selected from the groupconsisting of Schistosomiasis mansoni and S. japonica.
 8. A method forthe treatment of hepatic fibrosis in a subject, the hepatic fibrosisbeing caused by contact with a hepatotoxic chemical substance, themethod comprising the step of administering to the subject apharmaceutically effective amount of a compound having a formula:

wherein: R¹ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R² is a member ofthe group consisting of hydroxy, acetoxy and lower alkoxy, and R³ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2; and pharmaceutically acceptable salts thereof. 9.The method of claim 8, wherein said compound is Halofuginone.
 10. Themethod of claim 9, wherein said compound further includes apharmaceutically acceptable carrier.
 11. A method for the treatment ofan existing condition of hepatic cirrhosis in a subject, comprising thestep of administering to the subject a pharmaceutically effective amountof a compound having a formula:

wherein: R₁ is a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R₂ is a member ofthe group consisting of hydroxy, acetoxy and lower alkoxy, and R₃ is amember of the group consisting of hydrogen and lower alkenoxy-carbonyl;wherein n is 1 or 2; and pharmaceutically acceptable salts thereof. 12.The method of claim 11, wherein said compound is Halofuginone.
 13. Themethod of claim 11 wherein said compound further includes apharmaceutically acceptable carrier.